TLR4

In response to ligand binding to the Toll-like receptor 4 (TLR4) and myeloid differentiation-2 (MD-2) receptor complex, two major signaling pathways are activated that involve different adaptor proteins. One pathway depends on myeloid differentiation marker 88 (MyD88), which elicits proinflammatory responses, whereas the other depends on Toll-IL-1 receptor (TIR) domain-containing adaptor inducing interferon-β (TRIF), which elicits type I interferon production. Here, we showed that the TLR4 agonist and vaccine adjuvant CRX-547, a member of the aminoalkyl glucosaminide 4-phosphate (AGP) class of synthetic lipid A mimetics, displayed TRIF-selective signaling in human cells, which was dependent on a minor structural modification to the carboxyl bioisostere corresponding to the 1-phosphate group on most lipid A types. CRX-547 stimulated little or no activation of MyD88-dependent signaling molecules or cytokines, whereas its ability to activate the TRIF-dependent pathway was similar to that of a structurally related inflammatory AGP and of lipopolysaccharide from Salmonella minnesota. This TRIF-selective signaling response resulted in the production of substantially less of the proinflammatory mediators that are associated with MyD88 signaling, thereby potentially reducing toxicity and improving the therapeutic index of this synthetic TLR4 agonist and vaccine adjuvant [1].

For pharmacological analysis, human PBMC–derived monocytes and DCs were treated with a wide range of concentrations of CRX-527 or CRX-547, and the resulting dose-response curves were fit with a four-parameter logistic equation by means of XLFit software (IDBS) to determine ED50 (median effective dose or half-maximal effective dose) values and the extents of response. For inhibition experiments, cells were pretreated with constant concentrations of CRX-547, followed by a wide range of concentrations of CRX-527. To perform Schild regression, we determined an equi-active dose ratio (DR) for each inhibition curve by dividing the ED50 for the curve by the ED50 of the CRX-527–only response curve. Regression of log(DR-1) upon log[CRX-547] was used to fit a straight line with the intercept representing an estimate of the Kb for CRX-547. [1]

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Analysis of NF-κB nuclear translocation [1]
MonoMac6 cells in exponential growth in THP-1 medium were stimulated with increasing concentrations of CRX-527 or CRX-547 for the indicated times. Cells were fixed in 2% paraformalin overnight, treated with permeabilization buffer (PBS, 2% FBS, and 0.1% Triton X), and incubated with the following primary and secondary antibodies: antibody against NF-κB (p65), antibody against IRF3, fluorescein isothiocyanate (FITC)–conjugated antibody against rabbit antibody, and phycoerythrin (PE)–conjugated antibody against mouse antibody. Similarity scores of signaling protein localization with the nuclear stain DRAQ5 were used for ImageStream analysis of nuclear translocation with IDEAS software as previously described (Fig. 4A. Briefly, similarity scores were calculated as correlations between the pixel intensities of the nuclear (DRAQ5) and NF-κB images of stained cells that were hydrodynamically focused, excited by laser, and imaged on a charge-coupled device (CCD) camera. The similarity score is a derivation of Pearson’s correlation coefficient and achieves a higher positive value as the pixel intensities for NF-κB and nuclear staining become colocalized. A minimum of 3000 to 5000 cells were collected and analyzed for each condition and time point tested.

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Western blotting analysis [1]
For Western blotting analysis of total and phosphorylated proteins, primary human monocytes were isolated from PBMCs (1 × 107) by adherence to six-well culture plates for 2 hours at 37°C and in 5% CO2 and were treated with CRX-527 or CRX-547 (0.1 μM each) for the indicated times. After treatment, the cells were lysed with Cell Lysis Buffer containing Protease Inhibitor Cocktail. Proteins were resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. Membranes were incubated with primary monoclonal antibody against β-actin, antibody against pIRF3 (Ser396), primary polyclonal antibody against IRAK1, or antibody against IRF3 followed by incubation with horseradish peroxidase (HRP)–conjugated secondary antibodies against rabbit or mouse antibodies (KPL). Bands were detected with an ECL Advance Western Blot kit.

[1]. Selective TRIF-dependent signaling by a synthetic toll-like receptor 4 agonist. Sci Signal. 2012 Feb 14;5(211):ra13.

A similar selectivity for TRIF-dependent signaling in mice was reported for MLA. MLA lacks a phosphate on the reducing sugar at a similar position relative to the bioisosteric carboxyl of CRX-527 and CRX-547. The authors of these studies postulated that differences in MyD88- and TRIF-dependent gene expression downstream of TLR4 not only are related to differential activation of NF-κB and IRF3 but also may involve the differential activation of either phosphoinositide 3-kinase or p38 mitogen-activated protein kinase pathways. A recent study by Embry et al. suggests that a defect in IL-1β maturation after stimulation of TLR4 by sMLA is, in part, responsible for the reduced toxicity of MLA compared to that of LPS. Cells activated with sMLA are deficient in inducing the NLRP3 inflammasome (which is MyD88-dependent), as well as that of subsequent inflammasome assembly, caspase-1 activation, and release of mature IL-1β release. Comparison of the effects of synthetic sMLA with those of CRX-547 on human monocytes indicated that CRX-547 was TRIF-selective throughout the range of concentrations tested, whereas sMLA was TRIF-selective only at lower concentrations. Previous studies describing TRIF-dependent signaling by MLA have been conducted in murine systems. These data suggest that important signaling differences may exist for MLA and CRX-547 in human versus murine cells. Overall, our preliminary evaluation of mature IL-1β production by human PBMCs indicated that CRX-547 induced substantially less IL-1β than did CRX-527 (fig. S2), which is consistent with the inefficient activation by CRX-547 of the MyD88-dependent events that are needed for the inflammasome to function. [1]
The selective induction of TRIF-dependent signaling by synthetic lipid A mimetics suggests that further structure-activity investigations of these TLR4 agonists may provide opportunities for a better understanding of the underlying mechanisms that regulate TLR4 signaling pathways. The ability to selectively target either MyD88- or TRIF-dependent signaling downstream of TLR4 with synthetic receptor agonists may also provide valuable insights into the mechanism by which different lipid A structures differentially induce innate and adaptive immune responses. Such studies may be helpful in the development of synthetic vaccine adjuvants or new immunomodulators that selectively alter innate immune responses while simultaneously mitigating the potentially toxic side effects that are associated with the induction of inflammatory cytokines and chemokines. [1]

C81H151N2O19P

1488.068

1487.0649

C, 65.38; H, 10.23; N, 1.88; O, 20.43; P, 2.08

216014-05-0

216014-14-1 (CRX-527);216014-05-0 (CRX-547);

Typically exists as solid at room temperature

O(C(C[C@H](OC(CCCCCCCCC)=O)CCCCCCCCCCC)=O)[C@@H]1[C@@H](NC(C[C@H](OC(CCCCCCCCC)=O)CCCCCCCCCCC)=O)[C@H](OC[C@@H](NC(C[C@H](OC(CCCCCCCCC)=O)CCCCCCCCCCC)=O)C(O)=O)O[C@H](CO)[C@H]1OP(=O)(O)O

REEGNIYAMZUTIO-ARLDOHDRSA-N

InChI=1S/C81H151N2O19P/c1-7-13-19-25-31-34-40-43-49-55-66(97-73(87)58-52-46-37-28-22-16-10-4)61-71(85)82-69(80(91)92)65-96-81-77(83-72(86)62-67(56-50-44-41-35-32-26-20-14-8-2)98-74(88)59-53-47-38-29-23-17-11-5)79(78(70(64-84)100-81)102-103(93,94)95)101-76(90)63-68(57-51-45-42-36-33-27-21-15-9-3)99-75(89)60-54-48-39-30-24-18-12-6/h66-70,77-79,81,84H,7-65H2,1-6H3,(H,82,85)(H,83,86)(H,91,92)(H2,93,94,95)/t66-,67-,68-,69-,70-,77-,78-,79-,81-/m1/s1

Decanoic acid, (1R)-1-[2-[[(1R)-1-carboxy-2-[[2-deoxy-3-O-[(3R)-1-oxo-3-[(1-oxodecyl)oxy]tetradecyl]-2-[[(3R)-1-oxo-3-[(1-oxodecyl)oxy]tetradecyl]amino]-4-O-phosphono-β-D-glucopyranosyl]oxy]ethyl]amino]-2-oxoethyl]dodecyl ester (9CI, ACI)

CRX-547; CRX547; 216014-05-0; N-[(3R)-3-(Decanoyloxy)myristoyl]-O-[2-[[(3R)-3-(decanoyloxy)myristoyl]amino]-3-O-[(3R)-3-(decanoyloxy)myristoyl]-4-O-phosphono-2-deoxy-beta-D-glucopyranosyl]-D-serine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)